Methods and compositions for wound healing

ABSTRACT

Assays employing fibronectin-depleted substrates are described to identify invasion inducing agents. Such agents are useful for in vivo wound healing, including but not limited to deep wounds and chronic wounds.

This is a Continuation of application(s) Ser. No. 09/939,481 filed onAug. 24, 2001 now U.S. Pat No. 6,576,440, which is a Continuation ofapplication Ser. No. 09/503,998 filed on Feb. 14, 2000 which is issuedas U.S. Pat. No. 6,331,409 on Dec. 18, 2001, which is a Continuation ofapplication application Ser. No. 08/972,760 filed on Nov. 18, 1997 whichissued as U.S. Pat. No. 6,025,150 on Feb. 15, 2000, which is aContinuation-in-Part of application Ser. No. 08/754,322 filed on Nov.21, 1996, which issued as U.S. Pat. No. 5,840,514 on Nov. 24, 1998.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for woundhealing, and in particular, methods and compositions to promote andenhance wound healing.

BACKGROUND

The primary goal in the treatment of wounds is to achieve wound closure.Open cutaneous wounds represent one major category of wounds and includeburn wounds, neuropatic ulcers, pressure sores, venous stasis ulcers,and diabetic ulcers. Open cutaneous wounds routinely heal by a processwhich comprises six major components: i) inflammation, ii) fibroblastproliferation, iii) blood vessel proliferation, iv) connective tissuesynthesis v) epithelialization, and vi) wound contraction. Wound healingis impaired when these components, either individually or as a whole, donot function properly. Numerous factors can affect wound healing,including malnutrition, infection, pharmacological agents (e.g.,actinomycin and steroids), diabetes, and advanced age [see Hunt andGoodson in Current Surgical Diagnosis & Treatment (Way; Appleton &Lange), pp/86-98 (1988)].

With respect to diabetes, it is known that delayed wound healing causessubstantial morbidity in patients with diabetes. Diabetes mellitus is achronic disorder of glucose metabolism, and homeostasis that damagesmany organs. It is the eighth leading cause of death in the UnitedStates. M. Harris et al., “Prevalence of Diabetes and Impaired GlucoseTolerance and Glucose Levels in the US Population aged 20-40 Years,”Diabetes 36:523 (1987). In persons with diabetes, vascular disease,neuropathy, infections, and recurrent trauma predispose the extremities,especially the foot, to pathologic changes. These pathological changescan ultimately lead to chronic ulceration, which may necessitateamputation.

The most commonly used conventional modality to assist in wound healinginvolves the use of wound dressings. In the 1960s, a major breakthroughin wound care occurred when it was discovered that wound healing with amoist occlusive dressings was, generally speaking, more effective thanthe use of dry, non-occlusive dressings [Winter, Nature 193:293-94(1962)]. Today, numerous types of dressings are routinely used,including films (e.g., polyurethane films), hydrocolloids (hydrophiliccolloidal particles bound to polyurethane foam), hydrogels (cross-linkedpolymers containing about at least 60% water), foams (hydrophilic orhydrophobic), calcium alginates (nonwoven composites of fibers fromcalcium alginate), and cellophane (cellulose with a plasticizer) [Kannonand Garrett, Dermatol. Surg. 21:583-590 (1995); Davies, Burns 10:94(1983)]. Unfortunately, certain types of wounds (e.g., diabetic ulcers,pressure sores) and the wounds of certain subjects (e.g., recipients ofexogenous corticosteroids) do not heal in a timely manner (or at all)with the use of such dressings.

Several pharmaceutical modalities have also been utilized in an attemptto improve wound healing. For example, treatment regimens involving zincsulfate have been utilized by some practitioners. However, the efficacyof these regimens has been primarily attributed to their reversal of theeffects of sub-normal serum zinc levels (e.g., decreased host resistanceand altered intracellular bactericidal activity) [Riley, Am. Fam.Physician 24:107 (1981)]. While other vitamin and mineral deficiencieshave also been associated with decreased wound healing (e.g.,deficiencies of vitamins A, C and D; and calcium, magnesium, copper, andiron), there is no strong evidence that increasing the serum levels ofthese substances above their normal levels actually enhances woundhealing. Thus, except in very limited circumstances, the promotion ofwound healing with these agents has met with little success.

What is needed is a safe, effective, and interactive means for enhancingthe healing of wounds. The means should be able to be used withoutregard to the type of wound or the nature of the patient population towhich the subject belongs.

SUMMARY OF THE INVENTION

The present invention is directed at systems and methods for enhancingthe healing of wounds, especially chronic wounds (e.g., diabetic wounds,pressure sores). The compositions of the present invention are based onthe discovery that peptides containing the amino acid sequence PHSRN(SEQ ID NO:1) promote wound healing. The present invention contemplatesthe use of such peptides, peptide derivatives, protease-resistantpeptides, and non-peptide mimetics in the treatment of wounds.

It is not intended that the present invention be limited to the mode bywhich the compositions of the present invention are introduced to thepatient. In one embodiment, the present invention contemplates systemicadministration of the compound (e.g. intravenous). In anotherembodiment, the present invention contemplates topical administration,including but not limited to topical administration using solid supports(such as dressings and other matrices) and medicinal formulations (suchas mixtures, suspensions and ointments). In one embodiment, the solidsupport comprises a biocompatible membrane. In another embodiment, thesolid support comprises a wound dressing. In still another embodiment,the solid support comprises a band-aid.

The present invention contemplates a method for treating a wound,comprising a) providing: i) an invasion-inducing agent, and ii) asubject having at least one wound; and b) administering saidinvasion-inducing agent to said subject under conditions such that thehealing of said wound is promoted.

The present invention also contemplates a method for treating a wound,comprising a) providing: i) an invasion-inducing agent on a solidsupport, and ii) a subject having at least one wound; and b) placing thesolid support into the wound of the subject under conditions such thatthe healing of the wound is promoted.

The present invention also contemplates a method of screening candidateinvasion-inducing agents comprising: a) providing: i) inducible cells,ii) a fibronectin-depleted substrate, and iii) one or more candidateinvasion-inducing agents, b) contacting said cells in vitro with saidfibronectin-free substrate and said one or more candidateinvasion-inducing agents; and c) measuring the extent of cell invasionof said substrate. It is not intended that the present invention belimited to the type of inducible cells. In one embodiment, saidinducible cells are epithelial cells. In another embodiment, saidinducible cells are selected from the group consisting of fibroblasts,keratinocytes and muscle cells.

It is also not intended that the present invention be limited to aparticular invasion-inducing agent. In one embodiment, saidinvasion-inducing agent comprises a fibronectin-derived peptide. In apreferred embodiment, said peptide comprises the amino acid sequencePHSRN (SEQ ID NO:1). In yet another embodiment, said peptide lacks theRGD motif. In yet another embodiment, said peptide lacks the motif whichbinds the α5β1 receptor.

It is not intended that the present invention be limited by the lengthof the peptide. In one embodiment, said peptide is between five and fivehundred amino acids in length. In a preferred embodiment, said peptidecomprises the amino acids PHSRN (SEQ ID NO:1) and additional amino acidsadded to the amino terminus. In another embodiment, said peptidecomprises the amino acids PHSRN (SEQ ID NO:1) and additional amino acidsadded to the carboxy terminus. In yet another embodiment, said peptidescomprises the amino acids PHSRN (SEQ ID NO:1) and additional amino acidsadded to both the amino and carboxy termini.

It is not intended that the present invention be limited to specificinvasion-inducing agents. In one embodiment, the present inventioncontemplates invasion-inducing agents that comprise peptides that areprotease resistant. In one embodiment, such protease-resistant peptidesare peptides comprising protecting groups. In a preferred embodiment,endoprotease-resistance is achieved using peptides which comprise atleast one D-amino acid.

Definitions

To facilitate understanding of the invention set forth in the disclosurethat follows, a number of terms are defined below.

The term “wound” refers broadly to injuries to the skin and subcutaneoustissue initiated in different ways (e.g., pressure sores from extendedbed rest and wounds induced by trauma) and with varying characteristics.Wounds may be classified into one of four grades depending on the depthof the wound: i) Grade I: wounds limited to the epithelium; ii) GradeII: wounds extending into the dermis; iii) Grade III: wounds extendinginto the subcutaneous tissue; and iv) Grade IV (or full-thicknesswounds): wounds wherein bones are exposed (e.g., a bony pressure pointsuch as the greater trochanter or the sacrum). The term “partialthickness wound” refers to wounds that encompass Grades I-III; examplesof partial thickness wounds include burn wounds, pressure sores, venousstasis ulcers, and diabetic ulcers. The term “deep wound” is meant toinclude both Grade III and Grade IV wounds. The present inventioncontemplates treating all wound types, including deep wounds and chronicwounds.

The term “chronic wound” refers to a wound that has not healed within 30days.

The phrase “positioning the solid support in or on the wound” isintended to mean contacting some part of the wound with the solidsupport.

The phrases “promote wound healing,” “enhance wound healing,” and thelike refer to either the induction of the formation of granulationtissue of wound contraction and/or the induction of epithelialization(i.e., the generation of new cells in the epithelium). Wound healing isconveniently measured by decreasing wound area.

The phrase “wound fluid contents” refers to liquid associated with awound, as well as cells, cell factors, ions, macromolecules and proteinmaterial suspended such liquid at the wound site.

The term “subject” refers to both humans and animals.

The terms “enclosure,” “compartment,” and the like refer broadly to anycontainer capable of confining a solid support within a definedlocation.

The term “solid support” refers broadly to any support, including, butnot limited to, microcarrier beads, gels, Band-Aids™ and dressings.

The term “dressing” refers broadly to any material applied to a woundfor protection, absorbance, drainage, etc. Thus, adsorbent and absorbentmaterials are specifically contemplated as a solid support. Numeroustypes of dressings are commercially available, including films (e.g.,polyurethane films), hydrocolloids (hydrophilic colloidal particlesbound to polyurethane foam), hydrogels (cross-linked polymers containingabout at least 60% water), foams (hydrophilic or hydrophobic), calciumalginates (nonwoven composites of fibers from calcium alginate), andcellophane (cellulose with a plasticizer) [Kannon and Garrett, Dermatol.Surg. 21:583-590 (1995); Davies, Burns 10:94 (1983)]. The presentinvention specifically contemplates the use of dressings impregnatedwith the wound healing promoting and enhancing compounds of the presentinvention.

The term “biocompatible” means that there is minimal (i.e., nosignificant difference is seen compared to a control), if any, effect onthe surroundings. For example, in some embodiments of the presentinvention, the dressing comprises a biocompatible membrane.

The term “peptide derivative” refers to compound having an imino group(—NH—), and more particularly, a peptide bond. Peptides may be regaredas substituted amides. Like the amide group, the peptide bond shows ahigh degree of resonance stabilization. The C—N single bond in thepeptide linkage has typically about 40 percent double-bond character andthe C═O double bond about 40 percent single-bond character.

“Protecting groups” are those groups which prevent undesirable reactions(such as proteolysis) involving unprotected functional groups. In oneembodiment, the present invention contemplates that the protecting groupis an acyl or an amide. In one embodiment, the acyl is acetate. Inanother embodiment, the protecting group is a benzyl group. In anotherembodiment, the protecting group is a benzoyl group. The presentinvention also contemplates combinations of such protecting groups.

The term “Band-Aid™” is meant to indicate a relatively small adhesivestrip comprising and adsorbent pad (such as a gauze pad) for coveringminor wounds.

DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows the one embodiment of the substrate usedaccording to the present invention for testing invasion of cells (e.g.,fibroblasts). The spatial relationship of the ectoderm of theStrongylocentrotus purpuratus embryo to its extracellular matrix and toblastocoelar structures are shown (s, spicules; h, hyalin layer; e,ectoderm; b, subectodermal basement membrane; b1, blastocoel; g, stomachof the primitive gut; c, coelomic pouches). The esophagus and intestinedo not appear on the side of the embryo shown.

FIG. 2 is a graph showing the results of the testing of fibroblasts onfibronectin-depleted substrates in vitro with and withoutinvasion-inducing agents according one embodiment of the method of thepresent invention.

FIG. 3 is a graph showing the percentages of invaded neonatalfibroblasts, corresponding to various fragments of the plasmafibronectin cell binding domain, after placement on an invasionsubstrates. The 120 kDa and 39 kDa fragments contain the PHSRN (SEQ IDNO:1) sequence. The 11.5 kDa fragment does not. These fragments lack theα4β1 integrin binding site in the IIICS region.

FIG. 4 is a graph presenting a dose response curve relatingconcentration of peptides containing the amino acid sequence PHSRN (SEQID NO:1) to fibroblast invasion into an invasion substrate.

FIG. 5 is a graph presenting a dose response curve relatingconcentration of peptides containing the amino acid sequence PHSRN (SEQID NO:1) to keratinocyte invasion into an invasion substrate.

FIG. 6 is a graph presenting a dose response curve relatingconcentration of peptides containing the amino acid sequence PHSRN (SEQID NO:1) to human mammary or prostate epithelial cell invasion into aninvasion substrate.

FIG. 7 is a graph presenting the inductive effect of peptides containingthe amino acid sequence PHSRN (SEQ ID NO:1) on mouse muscle satellitecell invasion into an invasion substrate.

FIG. 8 is a graph presenting dermal wound closure data as a function oftime in genetically obese/diabetic mice, and their controls, in responseto treatment with peptides containing the amino acid sequence PHSRN (SEQID NO:1).

FIG. 9 is a graph presenting dermal wound closure data as a function oftime in diabetic and non-diabetic mice treated with peptides containingthe amino acid sequence PHSRN (SEQ ID NO:1).

FIG. 10 is a graph presenting the percentages of closed wounds as afunction of time in diabetic mice treated with peptides containing theamino acid sequence PHSRN (SEQ ID NO:1).

GENERAL DESCRIPTION OF THE INVENTION

The therapy of wounds, particularly those which are made difficult toheal by disease, has been attempted with a variety of purified growthfactors or cytokines because these molecules can induce cellularproliferation or increase the motility of cells in wounds. Thus, ifpresented in the correct form and location at the right time, growthfactors may greatly accelerate or enhance the healing of wounds bystimulating the growth of new tissue. Given the complexity and clinicalvariability of wounds, an obvious difficulty with the application ofspecific, purified growth factors or cytokines to wounded tissue, aloneor in combination, is that their forms or specific distributions in thewound may not support their normal activities. Instead, theeffectiveness of growth factors and cytokines in promoting the healingof wounded tissue may depend on their secretion by fibroblasts ormacrophages.

The present invention contemplates a more effective approach; thisapproach involves methods that stimulate the invasion of the wound bythe cells which synthesize the growth factors and cytokines active instimulating wound repair, especially monocytes, macrophages,keratinocytes, and fibroblasts. This strategy allows the cells in theirnormal in vivo setting to secrete the active factors. This approach hasa number of advantages: (1) the temporal and spatial distributions ofthe factors are likely to be optimal because the normally active cellsin their correct settings are secreting them; (2) all the appropriatefactors are likely to be present in their active forms, irrespective ofwhether they have been identified or cloned; (3) the sequential effectsof the factors in recruiting subsequent waves of cells involved in thehealing process to the wound site are likely to be enhanced by thepresence of more initiating cells in the wound. Thus, the presentinvention can be used to augment the effects of growth factor treatment.

The present invention is based on the discovery that the pure PHSRN (SEQID NO:1) peptide or purified plasma fibronectin fragments (i.e.,fibronectin-derived peptides) containing it, and lacking the α4β1integrin binding site in the IIICS region, are sufficient to stimulatefibroblast invasion of basement membranes in vitro under serum-freeconditions, while intact plasma fibronectin fails to stimulatefibroblast invasion. This suggests that this peptide, or forms of it notsubject to rapid proteolysis, may have similar effects on fibroblastsand monocytes/macrophages in vivo. Recruitment of fibroblasts ormonocytes/macrophages whose paracrine, regulatory effects on a varietyof neighboring cells are required for the early stages of wound healingis contemplated as a highly efficient and effective way to stimulate thecascade of regulatory interactions involved in wound healing becausethese cells will secrete the active factors or cytokines in the correcttemporal sequences and spatial locations to ensure their optimalactivities. PHSRN (SEQ ID NO: 1)-containing peptides (or structurallyrelated molecules) according to the present invention stimulate theentry of cells such as fibroblasts and monocyte/macrophages into theprovisional matrix of a wound, so that the entering cells themselvessecrete the factors and cytokines active in inducing or potentiatingwound healing.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention contemplates 1) assays to rapidly and readilyassess both a) the invasion potential of cells of patients (such as burnor diabetes patients) as well as b) the potential of candidate inducingagents (such as non-peptide compounds having similar activity to PHSRN(SEQ ID NO:1)-containing peptides) and 2) compositions and methods forthe treatment of patients with wounds.

A. Assays for Testing Invasion Potential and Screening New Therapeutics

It may be desirable to test the potential of cells for invasion, therebypredicting the ability of a patient to respond to the wound treatmentaccording to the present invention. Similarly, it may be desirable toscreen new potential therapeutics for their level of inducing activity.Two assay systems are contemplated for such testing.

1. Fibronectin-depleted Substrates

In one assay system, the present invention contemplates usingfibronectin-depleted substrates. These are substrates that originallycontain fibronectin that are treated according to the methods of thepresent invention (see below) to remove fibronectin. It is not intendedthat the present invention be limited by the nature of the originalsubstrate; such fibronectin-containing substrates suitable for treatmentand depletion include i) complex substrates containing a variety ofextracellular proteins and ii) less complex substrates containingfibronectin along with one or two other proteins (e.g., collagen,laminin, etc.).

It is also not intended that the present invention be limited by theprecise amount of fibronectin remaining after the substrate has beentreated. In other words, while the methods of the present inventionremove fibronectin, and in some embodiments, remove substantially allfibronectin, it is within the meaning of the term “fibronectin-depleted”substrate that a small amount of fibronectin remain in the substrate.

In one embodiment, the present invention contemplates using anextracellular matrix available commercially. For example, the presentinvention contemplates treating basement membrane matrices such as ECMGEL, a matrix from mouse sarcoma (commercially available from Sigma, St.Louis, Mo.). However, it is not intended that the present invention belimited by the particular fibronectin-containing substrate. For example,other commercially available substrates are contemplated, such as thecommonly used substrate Matrigel (available from Becton DickinsonLabware, Catalog #40234); Matrigel can be treated appropriatelyaccording to the methods of the present invention so as to render it“fibronectin-depleted” (see below).

Consequently, the present invention contemplates a fibronectin-freesubstrate. In this embodiment, Matrigel is treated so that it issubstantially fibronectin-free. The preparation of fibronectin-freeMatrigel involves “panning” the Matrigel substrate on gelatin as well as“panning” the substrate on anti-fibronectin antibody (anti-humanfibronectin IgG is available commercially, such as antibody from PromegaCorporation, Madison, Wis.).

2. Naturally Occurring Fibronectin-free Substrates

In another embodiment, the present invention contemplates substratesthat are naturally free of fibronectin; such a source provides, forexample, basement membranes permeable to select types of normallyinvasive cells, such membranes being naturally serum-free. In oneembodiment, the present invention contemplates sea urchins as a sourceof such membranes. In this regard, the ectoderm of sea urchin embryos isone cell thick, and secretes an underlying basement membrane (See,FIG. 1) very similar to that of mammals. These embryos contain nocirculatory or lymphatic systems; and thus, their basement membranes areserum-free. In embryos, the subectodermal basement membrane functionssimultaneously as a migration substrate for several, specificmesenchymal cell types while it functions as an invasion substrate forothers.

Sea urchin embryo basement membranes (SU-ECM) can be prepared by milddetergent treatment as described in D. Livant et al., Cancer Research55:5085 (1995). Briefly, adult Strongylocentrotus purpuratus sea urchinscan be obtained commercially ( e.g., from Pacific BioMarine), and theirembryos cultured to the early pluteus stage in artificial sea water at15° C. SU-ECM are then prepared from them by treatment with nonionicdetergent and strerilized by dilution in the appropriate media.

Cells for the invasion assay are harvested by rinsing in Hanks' balancedsalt solution, followed by brief treatment with 0.25% trypsin, 0.02%EDTA, and pelleting and resuspension in the appropriate medium with orwithout 5% FCS at a density of about 50,000 cells per ml. Whenappropriate, purified bovine plasma fibronectin (Sigma), purified 120kDa chymotryptic fragment (Gibco BRL) or PHSRN peptides (synthesized atthe Biomedical Research Core Facilities of the University of Michigan)are added to the resuspended cells prior to placement of the cells onSU-ECM. In each well of a plate used for an invasion assay, SU-ECM wereplaced in 0.5 ml of the appropriate medium, and 0.5 ml of theresuspended cells dropped on their exterior surfaces. Invasion assayswere incubated 1 to 16 hours prior to assay. If some circumstances,invasion assays were fixed in phosphate-buffered saline (PBS) with 2%formaldehyde for 5 minutes at room temperature, then rinsed into PBS.

Invasion assays are coded and scored blindly by microscopic examinationunder phase contrast at 200- and 400-fold magnification. Each cellcontacting an SU-ECM is scored for its position relative to the exterioror interior surfaces. A cell is judged to have invaded if it is locatedon an interior surface below the focal plane passing through the uppersurface of the SU-ECM, but above the focal plane passing through itslower surface. The minimum viability of the cells in each assay isalways ascertained at the time of assay by determining the fraction ofspread, adherent cells on the bottom of each well scored.

An invasion frequency is defined as the fraction of cells in contactwith basement membranes which are located in their interiors at the timeof assay. Thus, an invasion frequency of 1 denotes invasion by 100% ofthe cells in contact with basement membranes. Invasion frequencies aredetermined multiple times for each cell type assayed. For each type ofcell assayed the mean and standard deviation of the invasion frequencieswere calculated.

Regardless of which of the two types of substrates are employed, theinvasion substrates of the present invention are easy to prepare andgive rapid, highly consistent results with a variety of cells, includingbut not limited to fibroblasts and keratinocytes. While not limited toany mechanism, it is believed that cells exposed to invasion-inducingagents in this manner are potentially rendered capable of invading thesubstrate. Again, while not limited to any mechanism, it is believedthat the invasion inducing agent comprising the sequence PHSRN (SEQ IDNO:1) binds to the α5β1 receptor on the cell and thereby inducesinvasion of the substrate. In this regard, the present inventionprovides a method of treating cells comprising: a) providing i) cellsexpressing the α5β1 receptor, ii) a fibronectin-free substrate, and iii)one or more invasion-inducing agents; b) culturing said cells inserum-free culture media on said substrate in the presence of saidinvasion-inducing agents; and d) measuring the extent of cell invasionof said substrate. In one embodiment, the cells are human fibroblasts.

B. Compositions and Methods for the Treatment of Patients with Wounds

It is not intended that the present invention be limited by the natureof the wound healing promoting agent. Such agents can be identifiedfunctionally by simply testing them in the above-described in vitroassays. The extent of invasion of fibroblasts (or other suitable cells)with such agents is predictive of in vivo efficacy. Thus, the presentinvention contemplates in vivo treatment with “invasion-inducingagents,” i.e. those agents which have the capability of causing invasionof cells (such as fibroblasts and keratinocytes) in the above-describedin vitro assays.

1. Peptide Derivatives

In one embodiment, the invasion-inducing agent comprises a peptidederived from fibronectin. In a preferred embodiment, said peptidecomprises the sequence PHSRN (SEQ ID NO:1). Of course, the peptide maybe larger than five amino acids; indeed, the peptide fragment offibronectin may contain hundreds of additional residues (e.g., fivehundred amino acids). One such larger peptide is set forth in U.S. Pat.No. 5,492,890 (hereby incorporated by reference). In one embodiment, thePHSRN (SEQ ID NO:1)-containing peptide is less than one hundred aminoacids in length and lacks the RGD sequence characteristic offibronectin. A variety of PHSRN (SEQ ID NO:1)-containing peptides arecontemplated, including the PHSRN (SEQ ID NO:1) peptide itself andrelated peptides where additional amino acids are added to the carboxyterminus, including (but not limited to) peptides comprising thesequence: 1) PHSRN (SEQ ID NO:1), 2) PHSRNS (SEQ ID NO:3), 3) PHSRNSI(SEQ ID NO:4), 4) PHSRNSIT (SEQ ID NO:5), 5) PHSRNSITL (SEQ ID NO:6), 6)PHSRNSITLT (SEQ ID NO:7), 7) PHSRNSITLTN(SEQ ID NO:8), 8) PHSRNSITLTNL(SEQ ID NO:9), 9) PHSRNSITLTNLT (SEQ ID NO:10), 10) PHSRNSITLTNLTP (SEQID NO:11), and 11) PHSRNSITLTNLTPG(SEQ ID NO:12). Alternatively, PHSRN(SEQ ID NO:1)-containing peptides are contemplated where amino acids areadded to the amino terminus, including (but not limited to) peptidescomprising the sequence: 1) PEHFSGRPREDRVPHSRN (SEQ ID NO:13), 2)EHFSGRPREDRVPHSRN (SEQ ID NO:14), 3) HFSGRPREDRVPHSRN (SEQ ID NO:15), 4)FSGRPREDRVPHSRN (SEQ ID NO:16), 5) SGRPREDRVPHSRN (SEQ ID NO:17), 6)GRPREDRVPHSRN (SEQ ID NO:18), 7) RPREDRVPHSRN (SEQ ID NO:19), 8)PREDRVPHSRN (SEQ ID NO:20), 9) REDRVPHSRN (SEQ ID NO:21), 10) EDRVPHSRN(SEQ ID NO:22), 11) DRVPHSRN (SEQ ID NO:23), 12) RVPHSRN (SEQ ID NO:24),and 13) VPHSRN (SEQ ID NO:25). Finally, the present inventioncontemplates PHSRN (SEQ ID NO:1)-containing peptides where amino acidsare added to both the amino and carboxy termini, including (but notlimited to) peptides comprising the sequencePEHFSGRPREDRVPHSRNSITLTNLTPG (SEQ ID NO:26), as well as peptidescomprising portions or fragments of the PHSRN (SEQ ID NO: 1)-containingsequence PEHFSGRPREDRVPBSRNSITLTNLTPG (SEQ ID NO:27).

Peptides containing variations on the PHSRN (SEQ ID NO:1) motif arecontemplated. For example, the present invention also contemplates PPSRN(SEQ ID NO:85)-containing peptides for use in the above-named assays.Such peptides may vary in length in the manner described above for PHSRN(SEQ ID NO:1)-containing peptides. Alternatively, PPSRN (SEQ ID NO:85)may be used as a peptide of five amino acids.

Similarly, peptides comprising the sequence -HHSRN-(SEQ ID NO:28),-HPSRN-(SEQ ID NO:29), -PHTRN-(SEQ ID NO:30), -HHTRN-(SEQ ID NO:31),-HPTRN-(SEQ ID NO:32), -PHSNN-(SEQ ID NO:33), -HHSNN-(SEQ ID NO:34),-HPSNN-(SEQ ID NO:35), -PHTNN-(SEQ ID NO:36), -HHTNN-(SEQ ID NO:37),-HPTNN-(SEQ ID NO:38), -PHSKN-(SEQ ID NO:39), -HHSKN-(SEQ ID NO:40),-HPSKN-(SEQ ID NO:41), -PHTKN-(SEQ ID NO:42), -HHTKN-(SEQ ID NO:43),-HPTKN-(SEQ ID NO:44), -PHSRR-(SEQ ID NO:45), -HHSRR-(SEQ ID NO:46),-HPSRR-(SEQ ID NO:47), -PHTRR-(SEQ ID NO:48), -HHTRR-(SEQ ID NO:49),-HPTRR-(SEQ ID NO:50), -PHSNR-(SEQ ID NO:51), -HHSNR-(SEQ ID NO:52),-HPSNR-(SEQ ID NO:53), -PHTNR-(SEQ ID NO:54), -HHTNR-(SEQ ID NO:55),-HPTNR-(SEQ ID NO:56), -PHSKR-(SEQ ID NO:57), -HHSKR-(SEQ ID NO:58),-HPSKR-(SEQ ID NO:59), -PHTKR-(SEQ ID NO:60), -HHTKR-(SEQ ID NO:61),-HPTKR-(SEQ ID NO:62), -PHSRK-(SEQ ID NO:63), -HHSRK-(SEQ ID NO:64),-HPSRK-(SEQ ID NO:65), -PHTRK-(SEQ ID NO:66), -HHTRK-(SEQ ID NO:67),-HPTRK-(SEQ ID NO:68), -PHSNK-(SEQ ID NO:69), -HHSNK-(SEQ ID NO:70),-HPSNK-(SEQ ID NO:71), -PHTNK-(SEQ ID NO:72), -HHTNK-(SEQ ID NO:73),-HPTNK-(SEQ ID NO:74), -PHSKK-(SEQ ID NO:75), -HHSKK-(SEQ ID NO:76),-HPSKK-(SEQ ID NO:77), -PHTKK-(SEQ ID NO:78), -HRTKK-(SEQ ID NO:79), or-HPTKK-(SEQ ID NO:80) are contemplated by the present invention. Suchpeptides can be used as five amino acid peptides or can be part of alonger peptide (in the manner set forth above for PHSRN (SEQ IDNO:1)-containing peptides).

As noted above; the present invention contemplates peptides that areprotease resistant. In one embodiment, such protease-resistant peptidesare peptides comprising protecting groups. In a preferred embodiment,the contemplates a peptide containing the sequence PHSRN (SEQ IDNO:1)(or a variation as outlined above) that is protected fromexoproteinase degradation by N-terminal acetylation (“Ac”) andC-terminal amidation. The Ac-XPHSRNX-NH₂ (SEQ ID NO:84) peptide (whichmay or may not have additional amino acids, as represented by X; thenumber of additional amino acids may vary from between 0 and 100, ormore) is useful for in vivo administration because of its resistance toproteolysis.

In another embodiment, the present invention also contemplates peptidesprotected from endoprotease degradation by the substitution of L-aminoacids in said peptides with their corresponding D-isomers. It is notintended that the present invention be limited to particular amino acidsand particular D-isomers. This embodiment is feasible for all aminoacids, except glycine; that is to say, it is feasible for all aminoacids that have two stereoisomeric forms. By convention thesemirror-image structures are called the D and L forms of the amino acid.These forms cannot be interconverted without breaking a chemical bond.With rare exceptions, only the L forms of amino acids are found innaturally occurring proteins. In one embodiment, the present inventioncontemplates PHS(dR)N-containing peptides for wound healing

2. Mimetics

Compounds mimicking the necessary conformation for recognition anddocking to the receptor binding to the peptides of the present inventionare contemplated as within the scope of this invention. For example,mimetics of PHSRN (SEQ ID NO:1) peptides are contemplated. A variety ofdesigns for such mimetics are possible. For example, cyclic PHSRN (SEQID NO:1)-containing peptides, in which the necessary conformation forbinding is stabilized by nonpeptides, are specifically contemplated.U.S. Pat. No. 5,192,746 to Lobl, et al, U.S. Pat. No. 5,169,862 toBurke, Jr., et al, U.S. Pat. No. 5,539,085 to Bischoff, et al, U.S. Pat.No. 5,576,423 to Aversa, et al, U.S. Pat. No. 5,051,448 to Shashoua, andU.S. Pat. No. 5,559,103 to Gaeta, et al, all hereby incorporated byreference, describe multiple methods for creating such compounds.

Synthesis of nonpeptide compounds that mimic peptide sequences is alsoknown in the art. Eldred, et al, (J. Med. Chem. 37:3882 (1994)) describenonpeptide antagonists that mimic the Arg-Gly-Asp (SEQ ID NO:81)sequence. Likewise, Ku, et al, (J. Med. Chem. 38:9 (1995)) give furtherelucidation of the synthesis of a series of such compounds. Suchnonpeptide compounds that mimic PHSRN (SEQ ID NO:1) peptides arespecifically contemplated by the present invention.

The present invention also contemplates synthetic mimicking compoundsthat are multimeric compounds that repeat the relevant peptide sequence.In one embodiment of the present invention, it is contemplated that therelevant peptide sequence is Pro-His-Ser-Arg-Asn (SEQ ID NO:1) orPro-Pro-Ser-Arg-Asn (SEQ ID NO:27); in another embodiment, the relevantpeptide sequence is Ile-Lys-Val-Ala-Val (SEQ ID NO:2). As is known inthe art, peptides can be synthesized by linking an amino group to acarboxyl group that has been activated by reaction with a couplingagent, such as dicyclohexylcarbodiimide (DCC). The attack of a freeamino group on the activated carboxyl leads to the formation of apeptide bond and the release of dicyclohexylurea. It can be necessary toprotect potentially reactive groups other than the amino and carboxylgroups intended to react. For example, the α-amino group of thecomponent containing the activated carboxyl group can be blocked with atertbutyloxycarbonyl group. This protecting group can be subsequentlyremoved by exposing the peptide to dilute acid, which leaves peptidebonds intact.

With this method, peptides can be readily synthesized by a solid phasemethod by adding amino acids stepwise to a growing peptide chain that islinked to an insoluble matrix, such as polystyrene beads. Thecarboxyl-terminal amino acid (with an amino protecting group) of thedesired peptide sequence is first anchored to the polystyrene beads. Theprotecting group of the amino acid is then removed. The next amino acid(with the protecting group) is added with the coupling agent. This isfollowed by a washing cycle. The cycle is repeated as necessary.

In one embodiment, the mimetics of the present invention are peptideshaving sequence homology to the above-described PHSRN (SEQ IDNO:1)-containing peptides (including, but not limited to, peptides inwhich L-amino acids are replaced by their D-isomers). One commonmethodology for evaluating sequence homology, and more importantlystatistically significant similarities, is to use a Monte Carlo analysisusing an algorithm written by Lipman and Pearson to obtain a Z value.According to this analysis, a Z value greater than 6 indicates probablesignificance, and a Z value greater than 10 is considered to bestatistically significant. W. R. Pearson and D. J. Lipman, Proc. Natl.Acad. Sci. (USA), 85:2444-2448 (1988); D. J. Lipman and W. R. Pearson,Science, 227:1435-1441 (1985). In the present invention, syntheticpolypeptides useful in wound healing are those peptides withstatistically significant sequence homology and similarity (Z value ofLipman and Pearson algorithm in Monte Carlo analysis exceeding 6).

3. Formulations

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation, so long as thepreparation comprises an invasion-inducing agent. For example, suchcompositions can be provided together with physiologically tolerableliquid, gel or solid carriers, diluents, adjuvants and excipients.

These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts.

Such compositions are typically prepared as liquid solutions orsuspensions, or in solid forms. Formulations for wound healing usuallywill include such normally employed additives such as binders, fillers,carriers, preservatives, stabilizing agents, emulsifiers, buffers andexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, magnesiumcarbonate, and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%.

The compositions are also prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared.

The invasion-inducing agents of the present invention are often mixedwith diluents or excipients which are physiological tolerable andcompatible. Suitable diluents and excipients are, for example, water,saline, dextrose, glycerol, or the like, and combinations thereof. Inaddition, if desired the compositions may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, stabilizingor pH buffering agents.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases, suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides.

Experimental

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); μ (micron); M (Molar); μM(micromolar); mM (millimolar); N (Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nM (nanomolar); ° C. (degrees Centigrade); mAb(monoclonal antibody); MW (molecular weight); PBS (phophate bufferedsaline); U (units); d(days).

In some of the examples below, wounds are created in animals. Briefly,in one approach, experimental wounds were created in animals which werepre-anesthetized by inhalation of metofane and intradermal injection oflidocane. The hair on the backs of the animals was clipped and the skinwas disinfected with 70% ethanol. A piece of skin was then removed fromthe disinfected site with a 4 mm punch biopsy.

EXAMPLE 1 Production of Fibronectin-free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by affinity chromatography overGelatin-Sepharose (a technique which can be used to remove plasmafibronectin from fetal calf serum).

The Gelatin-Sepharose beads were obtained from Pharmacia(Catalog#170956-01). Two Kontes columns were set up with about 2 mls ofGelatin-Sepharose beads at 4 C to prevent gelling of the Matrigel. Thecolumns were then rinsed with about 10 column volumes of PBS to removethe preservative from the beads. The columns were drained to the top ofthe beads; then Matrigel was carefully added to the column. Once theMatrigel had entered the column, PBS was added to the top of the column.The Matrigel which was passed over the first column was collected andpassed over the second column. The fibronectin-depleted Matrigelcollected from the second column was plated on 48-well plates (150μl/well), sterilized under a UV light for 10 minutes and incubated at 37C overnight. The Matrigel treated in this manner failed to form a gel at37 C

EXAMPLE 2 Production of Fibronectin-free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by successive panning on gelatin.Eight wells of 24-well plate were coated with a 2% gelatin solution (thegelatin was obtained from Becton Dickinson Labware, Catalog #11868). Thewells were filled with the gelatin solution which had been heated to 50C and incubated for 3 minutes. Then the solution was removed and thewells were allowed to air dry. Following drying, the wells werethoroughly rinsed with H₂O followed by two rinses with PBS. The plateswere again allowed to dry; thereafter they were stored at −20 C untiluse. Matrigel was thawed on ice and then added to one of the wells of agelatin-coated plate (between 800 μl and 1 ml of Matrigel was added to awell of a 24-well plate). The plate was placed in a bucket of ice in a 4C room on an orbital shaker where the Matrigel was incubated in the wellfor two hours (although overnight incubation can be used). Following theincubation, the Matrigel was moved from the first well to a second welland then incubated for two hours under the same conditions. This processwas repeated until the Matrigel had been incubated on all eight wells ofthe gelatin-coated plate.

Following the depletion of the Matrigel, it was collected in Eppendorftubes. It was then plated on a 48-well plate 150 μl/well), sterilizedunder a UV light for 10 minutes and incubated at 37 C overnight. TheMatrigel formed as gel and the following day, cells were added to eachwell.

EXAMPLE 3 Production of Fibronectin-free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by gelatin panning followed byantibody panning.

-   Anti-fibronectin antibody-coated wells: Wells of a 24-well plate    were coated with an anti-fibronectin antibody. A mouse monoclonal    antibody to human fibronectin was obtained from Oncogene Science    (Catalog #CP13). Each well was incubated with 1 ml of antibody at a    concentration of 30 μl/ml for 2 hours at room temperature. Each well    was then incubated with a solution of 3% BSA in PBS for 2 hours at    room temperature. Following the two incubation periods, the wells    were thoroughly washed with PBS and stored at −20 C. until use.-   Depleting Matrigel of Fibronectin: Matrigel was panned over eight    gelatin-coated wells (as described above in Example 2) to remove    most of the fibronectin and its fragments. Thereafter, the Matrigel    was placed in the antibody-coated wells to remove any remaining    fragments of fibronectin which contain the cell-binding domain but    not the gelatin-binding domain. The Matrigel was incubated in an ice    bucket on an orbital shaker at 4 C for 2 hours. Once the Matrigel    was depleted, it was collected in Eppendorf tubes. The    fibronectin-depleted Matrigel was plated on a 48-well plate (150    μl/well), sterilized under a UV light for 10 minutes and incubated    at 37 C overnight. The Matrigel formed a gel and the following day,    cells were added to the wells.

EXAMPLE 4

In this example, normal, neonatal fibroblasts were tested on thedepleted Matrigel material prepared according to Example 3 above (i.e.antibody depletion). As shown in FIG. 2, panning with an antibody aftergelatin depletion improved the method for removal, as measured by thereduced invasiveness of fibroblasts. On the other hand, invasiveness ofthe fibroblasts could be induced by the addition of the PHSRN (SEQ IDNO:1) peptide. The success of antibody panning suggests the feasibilityof removing other components by the antibody panning methods. Otherserum components, such as thrombospondin, growth factors and cytokinesare contemplated by the present invention for removal by the appropriate(commercially available) antibody.

EXAMPLE 5 Conjugation of PHSRN (SEQ ID NO:1)-Containing Peptides

In this example, the preparation of a peptide conjugate is described.The synthetic peptide NH₂-PHSRNC (SEQ ID NO:82) can be preparedcommercially (e.g. Multiple Peptide Systems, San Diego, Calif.). Thecysteine is added to facilitate conjugation to other proteins. In orderto prepare a protein for conjugation (e.g. BSA), it is dissolved inbuffer (e.g., 0.01 M NaPO₄, pH 7.0) to a final concentration ofapproximately 20 mg/ml. At the same timen-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS” available fromPierce) is dissolved in N,N-dimethyl formamide to a concentration of 5mg/ml. The MBS solution, 0.51 ml, is added to 3.25 ml of the proteinsolution and incubated for 30 minutes at room temperature with stirringevery 5 minutes. The MBS-activated protein is then purified bychromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bed volume)equilibrated with 50 mM NaPO₄, pH 7.0 buffer. Peak fractions are pooled(6.0 ml).

The above-described cysteine-modified peptide (20 mg) is added to theactivated protein mixture, stirred until the peptide is dissolved andincubated 3 hours at room temperature. Within 20 minutes, the reactionmixture becomes cloudy and precipitates form. After 3 hours, thereaction mixture is centrifuged at 10,000×g for 10 min and thesupernatant analyzed for protein content. The conjugate precipitate iswashed three times with PBS and stored at 4° C.

EXAMPLE 6 Effect of Serum on PHSRN (SEQ ID NO:1) on Induction of HumanFibroblast Invasion

In this example, the invasiveness of neonatal fibroblasts into an SU-ECMinvasion substrate is considered. Experiments were performed underserum-free conditions, or in medium with 10% fetal calf serum (FCS).Neither serum-free medium nor medium containing serum supportedfibroblast invasion. However, consistent with the induction ofmetalloproteinase gene transcription by the 120 kDa fragment of plasmafibronectin (pFn) containing the cell-binding domain, the 120 kDafragment induced fibroblast invasion in the presence or in the absenceof serum.

To insure the induction of invasion documented in these experiments wasdue to pFn sequences, and not to bound growth factors or cytokines, allof the fragments used were purified by electrophoresis on denaturinggels, followed by electoelution. Also, all fragments and sequencestested here present in solution at a molar concentration equivalent tothat of plasma fibronectin in serum. The 120 kDa cell binding domainconsists of modules 2 through 11. Modules 9 and 10 are bound by the α5β1receptor because module 9 contains the PHSRN (SEQ ID NO:1) sequence,while module 10 has the RGD sequence. Accordingly, the invasion-inducingactivities of a gel-purified 39 kDa fragment containing modules 7-9 (andthe PHSRN (SEQ ID NO:1) sequence) with a gel-purified 11.5 kDa fragmentcontaining module 10 (and the RGD) sequence was considered. As can beseen in FIG. 3, all of the invasion-inducing activity of the plasmafibronectin cell-binding domain appeared to map to the 39 kDa fragmentbearing modules 7-9 and the PHSRN (SEQ ID NO:1) sequence. To test thisobservation rigorously, the PHSRN (SEQ ID NO:1) peptide, which wassynthesized in a peptide synthesis CORE facility, and the GRGDS (SEQ IDNO:83) peptide, which was obtained commercially, were tested in thepresence or in the absence of serum for their invasion-inducingactivities. As shown in FIG. 3, the PHSRN (SEQ ID NO:1) sequencecontained all the invasion-stimulatory activity of the pFn cell-bindingdomain; and the RGD sequence had no detectable activity at thenear-physiological concentrations used.

EXAMPLE 7

Dose-Response Effect Between PHSRN (SEQ ID NO:1) Concentration andFibroblast Invasion. In this example, fibroblasts were induced to invadeSU-ECM by concentrations of the PHSRN (SEQ ID NO:1) peptide ranging from10 to 3000 ng per ml in the presence, or in the absence of serum. As canbe seen from the dose response curves shown in FIG. 4, the PHSRN (SEQ IDNO:1) peptide was able to induce fibroblast invasion in the presence ofserum, which has been found to contain 40 to 80 micrograms per ml ofintact plasma fibronectin, and in its absence in a similar log-linearfashion. D. F. Mosher “Physiology of Fibronectin” Ann. Rev. Med. 35:561(1984).

These data suggest the metalloproteinase gene repressors produced byfibroblast α4β1 and α5β1 binding of intact plasma fibronectin do notappear to bind with such high affinity that they stop PHSRN (SEQ IDNO:1)-mediated invasion induction in the presence of serum. P. Huhtalaet al. “Cooperative Signaling by α5β1 and α4β1 Integrins RegulatesMetalloproteinase Gene Expression in Fibroblasts Adhering toFibronectin” J. Cell Biol. 129: 867 (1995). This observation isconsistent with the fact that, although induced by fibronectinfragments, fibroblast invasion in vivo must occur in the presence ofintact plasma fibronectin.

EXAMPLE 8 Induction of Keratinocyte Invasion by PHSRN (SEQ ID NO:1) inthe Presence of Serum

In this example the induction of normal keratinocyte invasion byPHSRN(SEQ ID NO:1) peptide, in the presence of serum, is presented.Normal neonatal keratinocytes were tested for their ability to beinduced to invade SU-ECM by the PHSRN (SEQ ID NO:1) peptide. It isnotable that the profile of keratinocyte invasive induction into SU-ECMby PHSRN (SEQ ID NO:1), presented in FIG. 5, is similar to the profileof invasive induction of fibroblast presented in Example 7. These datapresent the maximal invasion percentages for keratinocytes at a level ofabout 20%. Treatment of the cells (e.g., trypsin treatment) and assayconditions (e.g., time or orientation) are likely to effect this level.In any event, it is preferred that measurements are taken in the linearrange.

EXAMPLE 9 Invasiveness of Normal Human Mammary or Prostate EpithelialCell in Response to Induction By PHSRN (SEQ ID NO:1) in a SerumContaining Environment

In this example, data is presented, FIG. 6, demonstrating that PHSRN(SEQ ID NO:1) peptide also induces the invasive behaviors of humanmammary or prostate epithelial cells. These experiments were conductedin a serum containing environment using SU-ECM as an invasion substrate.As with fibroblasts, immunostaining experiments showed that mammary andprostate epithelial cells express both the α5β1 and the α4β1fibronectinreceptors (not shown); thus the ability of the α5β1 receptor to bind thePHSRN(SEQ ID NO:1) sequence on fibronectin fragments lacking the α4β1binding site, which are generated in wounds may induce these epithelialcells to migrate into the provisional matrix or into its adjacent stromato begin wound reepithelialization.

EXAMPLE 10 Invasiveness of Mouse Muscle Satellite Cells in Response toInduction by PHSRN (SEQ ID NO:1) in a Serum Containing Environment

In this example, the ability of the PHSRN (SEQ ID NO:1) peptide toinduce the invasive behavior of a third major tissue type, muscle cells,was considered. Mouse muscle satellite cells, which function as stemcells for muscle in vivo, were obtained from the laboratory of Dr. K.Kurachi (Department of Human Genetics). These cells were placed onSU-ECM invasion substrates in 1 microgram per ml of PHSRN (SEQ ID NO:1)peptide in the presence or absence of serum. As shown in FIG. 7, PHSRN(SEQ ID NO:1) induced the invasion of SU-ECM by muscle satellite cells.Since muscle satellite cells are normally located inside the basementmembranes surrounding the muscle fibers, in direct contact with musclecells, and since genetically engineered muscle cells have so far failedto cross the basement membranes separating them from the muscle fibersin vivo, it is interesting to speculate that treatment with the PHSRN(SEQ ID NO:1) invasion-inducing peptide may induce muscle satellite cellmigration into muscle in vivo, where these cells might resume normalfunction.

EXAMPLE 11 In Vivo Effect of PHSRN (SEQ ID NO:1)

In this example, the effect of PHSRN (SEQ ID NO:1) on a dermal woundcreated on genetically obese, diabetic mice was considered. FIG. 8presents the data from 10 mice wounded with 4 mm biopsy punch throughskin on the back. Just after wounding, the 5 treated mice receive 5microliters of normal saline containing 2 micrograms PHSRN (SEQ ID NO:1)peptide in their wounds. The 5 untreated mice received 5 microliters ofnormal saline without PHSRN (SEQ ID NO:1). Wound areas were measured onthe days indicated by standard procedures in which a microscope slide isplaced directly on the wound and its edges traced. The results show therate of wound closure was accelerated in the PHRSN (SEQ ID NO:1)-treatedgroup as compared to the untreated controls.

EXAMPLE 12 Comparative Wound Areas

In this example, the mean wound areas in PHSRN (SEQ ID NO:1)-treated anduntreated normal and diabetic mice are considered. With respect to thediabetic mice, ten obese diabetic C57B16Ksdb/db mice received dermalwounds with a biopsy punch on day 0 according to standard methods. Thewounds of 5 of these mice received 2 ug of the PHSRN (SEQ ID NO:1)peptide in 5 μl normal saline without peptide. On the days indicated,the edges of all wounds were traced onto glass slides and the areas ofthe tracings determined in square mm. This is a standard method forwound area measurement in these mice. Six tracings of every wound weredone on each day shown, and the mean wound areas determined.

With respect to non-diabetic littermates, eight non-diabetic C57B16Ksdb/+ mice received duplicate dermal wounds with a biopsy punch on day 0according to standard methods. 4 to 8 mm of unwounded skin separatedeach pair of wounds. One wound on each mouse received the PHSRN (SEQ IDNO:1) peptide as described above for the diabetic mice. The other woundreceived normal saline. On the days indicated, the edges of all woundswere traced onto glass slides for areas determination.

As shown in FIG. 9, diabetic (db/db) and normal (db/+) mice both presentratios which fall to zero. This means the treated wounds all closedprior to the untreated ones. If the PHSRN (SEQ ID NO:1) had no effect,the ratios of the wound areas should remain at about 1. In thealternative, if the PHSRN (SEQ ID NO:1) peptide slowed wound healingwith respect to untreated mice, the ratios should rise to infinity.Thus, a single application of the PHSRN (SEQ ID NO:1) peptide to thewound shortly after wounding dramatically stimulated wound healing inboth normal and diabetic mice.

It should also be noted that the rate of wound healing in the treatedcohort of mice, relative to the untreated cohort, is promotedapproximately equally in normal and diabetic mice through day four. Thisinterval corresponds to the time of provisional matrix induction (whichrequires invasion by fibroblasts, Leukocytes, and blood vessels). Itshould be noted from these data that in the later stages of woundhealing (after the first 4 days) the diabetic mice are less responsivethan the normal mice. These data are consistent with the hypothesis thatother late-occurring processes, which are PHSRN (SEQ IDNO:1)-independent, may still occur more slowly in diabetic mice ascompared to their normal littermates.

Expanding upon the data presented in FIG. 9, FIG. 10 presents thepercentages of completely closed wounds in PHSRN (SEQ ID NO:1)-treatedand untreated diabetic mice during the 41 day period after wounding bythe above described wounding methods. Again, the results show that asingle application of the PHSRN (SEQ ID NO:1) peptide to the woundshortly after wounding stimulated wound healing in both normal anddiabetic mice.

From the above, it should be evident that the present invention providesmethods and compositions for enhancing and promoting wound healing.Invasion-inducing agents can be readily identified using the assaysdescribed above. Thereafter, such agents can be modified or derivatizedand used therapeutically by application directly on wounds.

1. A peptide, comprising the amino acid sequence selected from the groupconsisting of PPSRN (SEQ ID NO:85), HHSRN (SEQ ID NO:28), and HPSRN (SEQID NO:29), wherein said peptide is protease resistant and comprisesprotecting groups.
 2. The peptide of claim 1, wherein said peptide isN-terminally acetylated and C-terminally amidated.
 3. The peptide ofclaim 1, wherein said peptide comprises at least one D-amino acid. 4.The peptide of claim 1, wherein said peptide is immobilized on a solidsupport.
 5. The peptide of claim 4, wherein said solid support comprisesa wound dressing.
 6. The peptide of claim 4, wherein said solid supportcomprises an adsorbent material.
 7. The peptide of claim 6, wherein saidadsorbent material is attached to an adhesive strip.